Total RNA purified with the RNeasy Midi Kit is of high quality and is suitable for many downstream applications. Protocols are also included for cleanup of partially purified RNA, in vitro transcripts, and RNA from enzymatic reactions. An additional buffer is required (composition provided) for isolation of cytoplasmic RNA from eukaryotic cells. Lyticase, zymolase, or glass beads (required for yeast samples) are not provided. Amounts of RNA isolated from samples can vary due to the developmental stage, species, and growth conditions of the sample source. Since the RNeasy procedure enriches for RNA species >200 nt, RNA yield does not include 5S rRNA, tRNAs, or other low-molecular-weight RNAs.|Formaldehyde agarose gel and northern blot of total RNA purified with the RNeasy Maxi Kit. Total RNA (10 µg) isolated from each source was loaded per lane. All tissues were from mouse. Yeast: Saccharomyces cerevisiae; E. coli strain: HB101. 32P-labeled probes recognized (G) GAPDH; (E) translation elongation factor EF-1α; and (O) outer membrane protein A sequences. (E and O were kindly provided by P. Philippsen, University of Basel, Switzerland and U. Henning, Max Planck Institute of Biology, Tübingen, Germany, respectively.) B. subtilis was not probed. M: 0.24–9.5 kb RNA ladder. 7.5 kb band (indicated) in embryo, Huh7, and HeLa cell lanes is a nuclear precursor RNA.|The RNeasy Midi spin column contained in the RNeasy Midi Kit.|

Total RNA purified with the RNeasy Midi Kit is of high quality and is suitable for many downstream applications (see figure "High-quality RNA from a variety of samples"). Total RNA is easily purified with the RNeasy Midi Kit from animal or human cells, animal or human tissues, and yeast (seetable).

Total RNA yields obtained with the RNeasy Midi Kit

Source

Starting material

Average yield (µg)

Animal cellsLMHHeLaCOS-7Lymphocytes (unstimulated)

7 x 1077 x 1073 x 1071 x 108

850100095050

Mouse tissueLiverLungSpleen

200 mg100 mg200 mg

700130600

Yeast cellsS. cerevisiae

2 x 108

450

Principle

The RNeasy Midi Kit allows efficient purification of total RNA from medium amounts of starting material. RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. RNeasy Kits provide the highest-quality RNA with minimum copurification of DNA.

Procedure

With the RNeasy Midi Kit, total RNA is easily purified from 5 x 106 to 1 x 108 animal or human cells, 20–250 mg animal or human tissues, and 2 x 107 to 5 x 108 yeast cells. Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane (see figure "RNeasy Midi spin column"). RNA binds (up to 1 mg capacity), and all contaminants are efficiently washed away. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment. Pure, concentrated RNA is eluted in 300–500 µl water (see figure "RNeasy Midi procedure"). A variety of special application protocols is also available.

Applications

RNA purified with RNeasy technology has A260/280 ratios of 1.9–2.1 (measured in 10 mM Tris·Cl, pH 7.5) and is ideal for use in all applications. Downstream applications include: